**5.1 Modifications of EV membrane with non-covalent and chemical modifications**

The role of EVs as encapsulated intercellular messengers makes them attractive for development into nanoscale therapeutic agents [91], and therefore, the need of augmenting the interaction with the recipient cell has been widely recognized. The higher rigidity of the membrane of EVs in comparison with their source cells does not appear to obstruct the efficient application of common hydrophobic insertion strategies to EVs. The introduction of small lipophilic ligands, such as membrane dyes, works effectively for their labeling and aids in monitoring in *in vitro* and *in vivo* experiments. Furthermore, hydrophobic loading is used for encapsulation of certain drugs and leads to their increased stability and therapeutic effect [92, 93].

Due to the negative charge on the recipient cells, binding and uptake of EVs were enhanced by increasing the charge interactions with an artificially introduced positive surface potential, as exemplified in their derivatization with cationic lipids [94]. A downside of this method can be that the extremely charged cationic reagents can cause cytotoxicity and the cellular uptake of the modified particles can proceed differently from the usual pathways, which results in an unpredictable cellular fate of the particle and its cargo and possibly its undesired degradation. The innate slightly negative electrostatic potential exhibited by unmodified EVs should contribute to the longer half-life of such particles *in vivo* as inferred from liposomal studies [95]. Further, the extremes of unbalanced positive charge may negatively impact the storage stability and the application of such reagents in high concentrations.

As an alternative to other methods, labeling of EV surfaces with fluorescent probes has been achieved using click chemistry without an apparent effect on the size and function of the particles [96]. Nevertheless, there is an immense complexity behind the engineering and downstream methods developed for other

**113**

**Figure 3.**

*Main engineering goals utilizing the modification of tetraspanins.*

*Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting*

on the source cells in combination with biotinylation *in vivo* [100].

immune response to tumor-associated antigens [102, 103].

*vivo* biodistribution studies [104].

biologicals conjugated with small molecules, especially when intended for thera-

Another example of employing an EV-localizing protein for efficient presentation on the EV surface involved a peptide or a protein fused with the C1C2 domain of lactadherin, which binds to EV outer membrane due to its affinity to phosphatidylserine, strongly enriched in exosome membranes. This method was used to generate antibodies against tumor biomarkers [101] and to increase the host

Strong binding of lactadherin to the exosome membrane was also the basis of an efficient labeling protocol utilizing overexpression of a fusion protein composed of *Gaussia* luciferase and a truncated lactadherin in source melanoma cells. After harvest by ultracentrifugation, the labeled exosomes were successfully used for *in* 

**5.2 Engineering of tetraspanin proteins for detection and monitoring of EVs**

Regarding the tetraspanins not only as very abundant, but as prominent structural and stability elements of the exosomal membrane, it is sensible to engineer genetic fusions of these proteins enriched in EVs to ensure a high density of the expressed product and optimize the derivatization for the different purposes of engineering: cognate ligand binding, labeling for visualization or isolation, modifying cargo uptake and transfer and stability (**Figure 3**). Most practically, transgene

The idea of decoration of EV membranes with functional ligands further led to the consideration of receptor binding strategies. Such EV functionalization can readily be achieved by modification of the source cell. A resulting opportunity harvesting the specificity of this approach could also be used as a strategy to eliminate vesicles implicated in pathological processes, such as cancer metastasis, or to neutralize the undesired activity of therapeutically applied vesicles. Transferrin-conjugated superparamagnetic nanoparticles, reactive with surface-expressed transferrin receptor of exosomes, enabled their isolation from blood and endowed the vesicles with superior targeting properties [99]. A robust labeling approach of microvesicles was the expression of biotin acceptor peptide-transmembrane domain (BAP-TM) receptor

*DOI: http://dx.doi.org/10.5772/intechopen.83537*

peutic purposes [97, 98].

*Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting DOI: http://dx.doi.org/10.5772/intechopen.83537*

biologicals conjugated with small molecules, especially when intended for therapeutic purposes [97, 98].

The idea of decoration of EV membranes with functional ligands further led to the consideration of receptor binding strategies. Such EV functionalization can readily be achieved by modification of the source cell. A resulting opportunity harvesting the specificity of this approach could also be used as a strategy to eliminate vesicles implicated in pathological processes, such as cancer metastasis, or to neutralize the undesired activity of therapeutically applied vesicles. Transferrin-conjugated superparamagnetic nanoparticles, reactive with surface-expressed transferrin receptor of exosomes, enabled their isolation from blood and endowed the vesicles with superior targeting properties [99]. A robust labeling approach of microvesicles was the expression of biotin acceptor peptide-transmembrane domain (BAP-TM) receptor on the source cells in combination with biotinylation *in vivo* [100].

Another example of employing an EV-localizing protein for efficient presentation on the EV surface involved a peptide or a protein fused with the C1C2 domain of lactadherin, which binds to EV outer membrane due to its affinity to phosphatidylserine, strongly enriched in exosome membranes. This method was used to generate antibodies against tumor biomarkers [101] and to increase the host immune response to tumor-associated antigens [102, 103].

Strong binding of lactadherin to the exosome membrane was also the basis of an efficient labeling protocol utilizing overexpression of a fusion protein composed of *Gaussia* luciferase and a truncated lactadherin in source melanoma cells. After harvest by ultracentrifugation, the labeled exosomes were successfully used for *in vivo* biodistribution studies [104].

#### **5.2 Engineering of tetraspanin proteins for detection and monitoring of EVs**

Regarding the tetraspanins not only as very abundant, but as prominent structural and stability elements of the exosomal membrane, it is sensible to engineer genetic fusions of these proteins enriched in EVs to ensure a high density of the expressed product and optimize the derivatization for the different purposes of engineering: cognate ligand binding, labeling for visualization or isolation, modifying cargo uptake and transfer and stability (**Figure 3**). Most practically, transgene

**Figure 3.** *Main engineering goals utilizing the modification of tetraspanins.*

*Current Topics in Biochemical Engineering*

**4.2 Mechanisms of EV entry into the target cell**

cally acting epidermal growth factor [90].

**cells**

**modifications**

When an EV attaches to the target cell surface, it can in some cases activate the cognate receptors without internalization or transfer of the content to the recipient cell via its fusion with the target cell membrane or via endocytosis [84]. Endocytosis is an active process that requires cytoskeletal remodeling dependent on actin dynamics and includes clathrin-dependent endocytosis, phagocytosis and macropinocytosis. The clathrin-dependent endocytosis has been established as cellular entry for EVs based on the experiments with specific inhibitors of this pathway. Additionally, endocytic uptake of EVs can involve lipid rafts, sometimes dependent on caveolin proteins. The size of EVs may be a limiting factor for cellular entry via endocytosis [85]. The EV uptake by phagocytosis was monitored by their high level of accumulation in phagocytic cells and localization into the phagolysosome [86], as well as the identification of the crucial role of the phosphatidylserine binding T-cell immunoglobulin and mucin domain containing (TIM4) receptor for the uptake of exosomes into macrophages [87, 88]. The contribution of macropinocytosis pathway was revealed with studies where exosomal uptake was decreased by the inhibition of cytoskeletal rearrangements that normally lead to membrane ruffles [89], as well as with its promotion caused by the activation of the agonisti-

**5. Molecular engineering to facilitate EV labeling and delivery to target** 

The role of EVs as encapsulated intercellular messengers makes them attractive for development into nanoscale therapeutic agents [91], and therefore, the need of augmenting the interaction with the recipient cell has been widely recognized. The higher rigidity of the membrane of EVs in comparison with their source cells does not appear to obstruct the efficient application of common hydrophobic insertion strategies to EVs. The introduction of small lipophilic ligands, such as membrane dyes, works effectively for their labeling and aids in monitoring in *in vitro* and *in vivo* experiments. Furthermore, hydrophobic loading is used for encapsulation of certain drugs and leads to their increased stability and therapeutic effect [92, 93]. Due to the negative charge on the recipient cells, binding and uptake of EVs were enhanced by increasing the charge interactions with an artificially introduced positive surface potential, as exemplified in their derivatization with cationic lipids [94]. A downside of this method can be that the extremely charged cationic reagents can cause cytotoxicity and the cellular uptake of the modified particles can proceed differently from the usual pathways, which results in an unpredictable cellular fate of the particle and its cargo and possibly its undesired degradation. The innate slightly negative electrostatic potential exhibited by unmodified EVs should contribute to the longer half-life of such particles *in vivo* as inferred from liposomal studies [95]. Further, the extremes of unbalanced positive charge may negatively impact the storage stability and the application of such reagents in high

As an alternative to other methods, labeling of EV surfaces with fluorescent probes has been achieved using click chemistry without an apparent effect on the size and function of the particles [96]. Nevertheless, there is an immense complexity behind the engineering and downstream methods developed for other

**5.1 Modifications of EV membrane with non-covalent and chemical** 

**112**

concentrations.

expression in the parent cell would be induced to deliver EVs enriched in the modified protein. The success of such set-up depends on the understanding of both molecular biology and protein architecture of the targeted species.

In one of the pioneering studies, certain sites on the tetraspanin CD63 have been chosen to allow the integration of fluorescent fusion proteins on the extraand intravesicular side of the exosomal membrane [105]. CD63-GFP fusions have proven valuable reporters in the elucidation of the role of immune synapse in secretion of exosomes from T-cells to APCs and their fusion with recipient cells [106], in determining differential uptake properties of different immune cell subsets for EVs originating from a cancer cell line [107] and in an *in vivo* study imaging the fate of EVs produced by breast cancer cell line in *nude* mice [108].

## **5.3 Engineering of EV surface proteins for enhanced cell-type specific targeting: introduction of target specificity, stability and improved cargo delivery**

In the pioneering example of derivatization of a protein enriched in exosomal membrane to achieve specific targeting [109], the rabies virus glycoprotein (RVG) peptide fused with lysosome-associated membrane glycoprotein 2b (Lamp-2b) was used to target exosomes to the central nervous system in an *in vivo* mouse model. Immature dendritic cells-derived exosomes, enriched in an N-terminal fusion of an αv integrin-specific internalizing RGD-sequence containing peptide with this scaffold, could internalize efficiently into target-positive breast cancer cells [110]. The display of such constructs could be efficiently enhanced by introducing a protective glycosylation motive, which improved the surface expression of exosome-bound N-terminally fused peptides by preventing their acid-mediated proteolysis during endosomal passage and indeed led to a more efficient specific cellular uptake of exosomes engineered in this way [111]. Specific targeting of IL3-receptor overexpressing chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells has been shown for exosomes armed with a fusion protein of IL3 and Lamp2b and loaded with Imatinib, a tyrosine kinase inhibitor, and led to a the reduction of tumor size *in vivo* [112]. Exosomes engineered in this way achieved a higher abundance at the tumor site and were hence able to inhibit xenograft growth more efficiently than the active ingredient alone or than loaded control exosomes.

Glycosylphosphatidylinositol (GPI)-mediated anchoring of the specific targeting unit was demonstrated to be a stable alternative to Lamp2b fusions. EVs expressing GPI-anchored nanobodies, specific to epidermal growth factor receptor (EGFR), displayed enhanced binding to the EGFR-overexpressing cancer cells. This, however, did not lead to an increased uptake, and it was suggested that in this particular biological system not only the affinity but also the density of the targeting ligand must be high enough to induce receptor clustering and subsequent internalization [113]. Exosomes derived from a HEK cell line transfected with a construct of platelet-derived growth factor (PDGF)-anchoring sequence and an EGFR-binding peptide were on the other hand efficient in targeting EGFRexpressing tumors *in vivo* and reducing their size with delivery of microRNA Let7 [114]. Enhanced uptake has been achieved for EVs enriched in a fusion protein of tetraspanin CD63 and stearylated octaarginine, a representative cell-penetrating peptide, by their ability to induce active macropinocytosis [115].

The concept of enhancing the stability of the targeted exosomal surface protein intended for fusion with a targeting agent to allow a higher degree of versatility for their modification for an improved target recognition has raised interest in their engineering at the protein level. A significant increase in thermal stability has been achieved by introduction of additional disulfide bonds in the EC2 of tetraspanin CD81, with the best variant exhibiting a positive shift in the melting temperature

**115**

*Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting*

evolution of tetraspanin proteins towards novel antigen binding.

for 45°C comparing with the wild-type protein [116]. When engrafted with a human transferrin-receptor specific peptide, the stabilized scaffold exhibited significantly better biophysical properties than the analogously engrafted wild-type protein. In the same study, a mutant has been discovered that exhibits reversible unfolding behavior up to a temperature of 110°C in contrast to the wild-type CD81 EC2, which presents another option for extensive engineering required for directed

Not only can the overexpression of tetraspanins in source cell lines increase the production and stability of exosomes, but also the exosomes engineered to contain adeno-associated viruses (AAVs), designed for improved delivery of genetic material to target cell [100], were superior in their yield and functionality when CD9 was overexpressed in AAV-producer cells [10]. CD9 overexpression has also increased the speed and transduction efficiency of lentiviral gene delivery into numerous cell lines, confirming the important role of this tetraspanin in gene

The current scope of EV engineering reaches beyond receptor targeting systems and aspires towards modifications with complex modules, assigning them simultaneously with multiple novel functionalities, such as specific recognition as well as enzymatic activity, to enhance their potential therapeutic effect. Recently, regression of orthotopic Her2-positive tumors has been achieved by applying exosomes, able of specific targeting via their surface decoration with a fusion protein of high-affinity anti-Her2 single-chain Fv, and containing mRNA encoding a bacterial enzyme Hchr6, which in the strongly Her2-positive cells catalyzed the conversion of the prodrug CNOB to cytotoxic MCHB [118]. Tetraspanin engineering could support a sophisticated concept to aid intracellular delivery of exosomal cargo proteins, directionally incorporated during vesicle biogenesis [119]. Genes encoding two recombinant proteins, a fusion of photoreceptor cryptochrome 2 (CRY2) and the protein of interest and a fusion of tetraspanin protein CD9 and CRY-interacting basic-helix–loop–helix 1 (CIB1), were co-transfected into a single cell line. The blue light-induced binding of CRY2 and CIB1 enabled docking of CRY2-fused target proteins into nascent exosomes, and in the absence of the blue light, the cargo protein was released into the exosomal lumen. Transfer of Cre recombinase confirmed

The tetraspanins, well established as the biomarker proteins of extracellular vesicles, have been addressed for increasing EV stability and improving their function as delivery vehicles, both by assigning them with target recognition properties and modulating their cargo transfer. From the current point of view, the complexity of the tetraspanin-mediated interactions and signaling networks formed in a cell is yet to be discerned. Tetraspanins are known to interact naturally with a plethora of cell surface-bound ligands, which results in potent biological effects conveyed through different pathways; however, systematic evaluation of the affinity of the association with the interaction partners would assist in prediction of the consequential cellular processes as well as in determining optimal choice of the tetraspanin targeted for modification. There are recent reports describing modified tetraspanins mediating both surface protein interactions and an intracellular fusion-mediated enzymatic activity, which underline the feasibility of engineering versatile functions into tetraspanin proteins as fusion partners. The structural details on tetraspanins modified in this way, as well as the read-out revealing their actual behavior in the foreseen role and the influence of such modifications on

*DOI: http://dx.doi.org/10.5772/intechopen.83537*

the efficiency of this system *in vitro* and *in vivo*.

transfer [117].

**6. Conclusions**

#### *Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting DOI: http://dx.doi.org/10.5772/intechopen.83537*

for 45°C comparing with the wild-type protein [116]. When engrafted with a human transferrin-receptor specific peptide, the stabilized scaffold exhibited significantly better biophysical properties than the analogously engrafted wild-type protein. In the same study, a mutant has been discovered that exhibits reversible unfolding behavior up to a temperature of 110°C in contrast to the wild-type CD81 EC2, which presents another option for extensive engineering required for directed evolution of tetraspanin proteins towards novel antigen binding.

Not only can the overexpression of tetraspanins in source cell lines increase the production and stability of exosomes, but also the exosomes engineered to contain adeno-associated viruses (AAVs), designed for improved delivery of genetic material to target cell [100], were superior in their yield and functionality when CD9 was overexpressed in AAV-producer cells [10]. CD9 overexpression has also increased the speed and transduction efficiency of lentiviral gene delivery into numerous cell lines, confirming the important role of this tetraspanin in gene transfer [117].

The current scope of EV engineering reaches beyond receptor targeting systems and aspires towards modifications with complex modules, assigning them simultaneously with multiple novel functionalities, such as specific recognition as well as enzymatic activity, to enhance their potential therapeutic effect. Recently, regression of orthotopic Her2-positive tumors has been achieved by applying exosomes, able of specific targeting via their surface decoration with a fusion protein of high-affinity anti-Her2 single-chain Fv, and containing mRNA encoding a bacterial enzyme Hchr6, which in the strongly Her2-positive cells catalyzed the conversion of the prodrug CNOB to cytotoxic MCHB [118]. Tetraspanin engineering could support a sophisticated concept to aid intracellular delivery of exosomal cargo proteins, directionally incorporated during vesicle biogenesis [119]. Genes encoding two recombinant proteins, a fusion of photoreceptor cryptochrome 2 (CRY2) and the protein of interest and a fusion of tetraspanin protein CD9 and CRY-interacting basic-helix–loop–helix 1 (CIB1), were co-transfected into a single cell line. The blue light-induced binding of CRY2 and CIB1 enabled docking of CRY2-fused target proteins into nascent exosomes, and in the absence of the blue light, the cargo protein was released into the exosomal lumen. Transfer of Cre recombinase confirmed the efficiency of this system *in vitro* and *in vivo*.
